The OVERALL OBJECTIVES of this project are to understand the altered regulation of microRNAs by oncogenic signals in cholangiocarcinoma, and how microRNAs contribute to cell death resistance. Approaches that subvert survival pathways have demonstrated that tumor cells are not endowed with impenetrable armor, but are rather fragile, and dependent on ongoing prosurvival mechanisms. Recently, an RNA-based layer of gene regulation has emerged that promotes tumor cell survival, with a prominent role for microRNAs. MicroRNAs act to fine tune the proteome in a cell-type- and stimulus-specific manner and altered microRNA expression is a feature of human cancers. Sonic Hedgehog, a developmental pathway reactivated in cholangiocarcinoma, may play a role in microRNA alterations. Preliminary studies investigating microRNA targets of Hedgehog signaling have identified three microRNAs, mir-106b, mir-93, and mir-25 that are dependent upon Hedgehog for expression. These microRNAs are clustered on chromosome 7 and collectively referred to as mir-106b~mir-25. Additional preliminary data using computational analysis and cell culture models has confirmed that mir-25 protects cells from TRAIL-induced apoptosis by repressing expression of Death Receptor-4 (one of two pro-apoptotic TRAIL receptors) as well as the pro-apoptotic BH3 protein Bim. Based on these observations, the CENTRAL HYPOTHESIS of this proposal is that Hedgehog signaling protects against TRAIL-induced death via increased expression of mir-106b~mir-25 and reciprocal silencing of key apoptotic signaling polypeptides, DR4 and Bim. The SPECIFIC AIMS are to test the hypotheses that: (1) Hedgehog signaling, by MCM7 host-gene transcription, stimulates mir-106b~mir-25 production;and (2) mir-25, mir-93, and mir-106b protect cells from apoptosis by directly targeting Death Receptor-4 and Bim mRNAs. Advanced molecular and cellular tools to manipulate microRNAs and Hedgehog signaling have been developed to address these questions of biomedical importance in hepatobiliary neoplasia. The current application seeks to advance the scientific knowledge regarding cell death signaling in cholangiocarcinoma. Thus, the results of the proposed experiments are related to improving health by understanding the altered pathways promoting cell death resistance in liver cancer. Successful completion of this proposal will provide needed mechanistic insight to influence clinical approaches to compel cholangiocarcinoma cell death through Hedgehog inhibition.